The present invention is directed to a novel method of performing hot start PCR reactions. Furthermore, the present invention relates to achieving a greater specificity of amplification of a target nucleic acid. Also provided in the present invention are reagents and kits for performing magnesium precipitate hot start PCR reactions.
PCR is a rapid and simple method for specifically amplifying a target DNA sequence in an exponential manner. Saiki, et al., Science 239:487-4391 (1988). Briefly, the method as now commonly practiced utilizes a pair of primers that have nucleotide sequences complementary to the DNA which flanks the target sequence. The primers are mixed with a solution containing the target DNA (the template), a thermostable DNA polymerase and deoxynucleoside triphosphates (dNTPS) for all four deoxynucleotides (adenosine (A), tyrosine (T), cytosine (C) and guanine(G)). The mix is then heated to a temperature sufficient to separate the two complementary strands of DNA. The mix is next cooled to a temperature sufficient to allow the primers to specifically anneal to sequences flanking the gene or sequence of interest. The temperature of the reaction mixture is then optionally reset to the optimum for the thermostable DNA polymerase to allow DNA synthesis (extension) to proceed. The temperature regimen is then repeated to constitute each amplification cycle. Thus, PCR consists of multiple cycles of DNA melting, annealing and extension. Twenty replication cycles can yield up to a million-fold amplification of the target DNA sequence. In some applications a single primer sequence functions to prime at both ends of the target, but this only works efficiently if the primer is not too long in length. In some applications several pairs of primers are employed in a process commonly known as multiplex PCR.
The ability to amplify a target DNA molecule by PCR has applications in various areas of technology e.g., environmental and food microbiology (Wernars et al., Appl. Env. Microbiol., 57:1914-1919 (1991); Hill and Keasler, Int. J. Food Microbiol., 12:67-75 (1991)), clinical microbiology (Wages et al. J. Med. Virol., 33:58-63 (1991); Sacramento et al., Mol. Cell Probes, 5:229-240 (1991)), oncology (Kumar and Barbacid, Oncogene, 3:647-651 (1988); McCormick, Cancer Cells, 1:56-61 (1989)), genetic disease prognosis (Handyside et al., Nature, 344:768-770 (1990)), and blood banking and forensics (Jackson, Transfusion, 30:51-57 (1990)).
Although significant progress has been made in PCR technology, the amplification of non-target oligonucleotides due to side-reactions, such as mispriming on non-target background DNA, RNA, and/or the primers themselves, still presents a significant problem. This is especially true in diagnostic applications where PCR is carried out in a milieu containing complex background DNA and/or RNA while the target DNA may be present at a very low level down to a single copy (Chou et al., Nucleic Acid Res., 20:1717-1723 (1992)).
The temperature at which Taq DNA polymerase exhibits highest activity is in the range of 62xc2x0 to 72xc2x0 C., however, significant activity is also exhibited in the range of 20xc2x0 to 37xc2x0 C. As a result, during standard PCR preparation at ambient temperatures, the primers may prime DNA extension at non-specific sequences because the formation of only a few base pairs at the 3xe2x80x2-end of a primer can result in a stable priming complex. The result can be competitive or inhibitory products at the expense of the desired product. As an example of inhibitory product, structures consisting only of primer, sometimes called xe2x80x9cprimer dimersxe2x80x9d are formed by the action of DNA polymerase on primers paired with each other, regardless of the true target template. The probability of undesirable primer-primer interactions increases with the number of primer pairs in the reaction, as with multiplex PCR. Other examples of inhibitory products are xe2x80x9cwrong bandsxe2x80x9d of various length, caused by mispriming on the template DNA. During PCR cycling, these non-specific extension products can compete with the desired target DNA and/or lead to misinterpretation of the assay.
Since these side reactions often occur during standard PCR preparation at ambient temperature, one method for minimizing these side reactions involves xe2x80x9chot startxe2x80x9d PCR. Many PCR analyses, particularly the most demanding ones, benefit from a hot start. About 50% of all PCR reactions show improved yield and/or specificity if a hot start is employed, and in some cases a hot start is absolutely critical. These demanding PCR analyses include those which have very low copy numbers of target (such as 1 HIV genome per 10,000 cells), denatured DNA (many DNA extraction procedures include a boiling step, so that the template is single-stranded during reaction setup), or contaminated DNA e.g., DNA from soil or feces and/or DNA containing large amounts of RNA. However, current methods of achieving a hot start are tedious, expensive, and/or have other shortcomings.
Hot start PCR may be accomplished by various physical, chemical, or biochemical methods. In a physical hot start, the DNA polymerase or one or more reaction components that are essential for DNA polymerase activity is not allowed to contact the sample DNA until all the components required for the reaction are at a high temperature. The temperature must be high enough so that not even partial hybridization of the primers can occur at any locations other than the desired template location, in spite of the entire genome of the cell being available for non-specific partial hybridization of the primers. Thus, the temperature must be high enough so that base pairing of the primers cannot occur at template (or contaminating template) locations with less than perfect or near-perfect homology. This safe starting temperature is typically in the range of 50xc2x0 to 75xc2x0 C. and typically is about 10xc2x0 C. hotter than the annealing temperature used in the PCR.
One physical way a hot start can be achieved is by using a wax barrier, such as the method disclosed in U.S. Pat. Nos. 5,599,660 and 5,411,876. See also Hebert et al., Mol. Cell Probes, 7:249-252 (1993); Horton et al., Biotechniques, 16:42-43 (1994). Using such methods, the PCR reaction is set up in two layers separated by a 1 mm thick layer of paraffin wax which melts at about 56xc2x0 C. There are several methods which may be used to separate the reaction components into two solutions. For instance, all of the DNA is added, with 1xc3x97buffer but no dNTPs and no DNA polymerase enzyme, in a volume of 25 ml. One drop of melted wax is added and the tubes are all heated to 60xc2x0 C. for one minute to allow the melted wax to form a sealing layer after which the tubes are cooled so the wax solidifies. Then a 25 ml mixture containing 1xc3x97buffer, all of the dNTPs, and the enzyme is added to each reaction. Finally, 1 drop of oil is added, to make 4 total layers. As the thermal cycler protocol heats the tubes to the first melting step (approximately 95xc2x0 C.), the wax melts and floats to mix with the oil layer, and the two aqueous layers mix by convection as the temperature cycles.
One common variation involving the use of a wax barrier is that the reaction components are assembled with no magnesium ions so that the DNA polymerase enzyme is inactive. The magnesium ion encased in a wax bead is then (or initially) added. A further modification of the wax barrier used in PCR reactions is disclosed in the U.S. Pat. No. 5,599,660. Alternatively, at least one biological or chemical reagent needed for PCR is mixed with a wax carrier, resulting in a reagent that is solid at room temperature. Thus, the addition of other PCR reagents does not activate the DNA polymerase due to the fact that one or some of the reagents are sequestered in the wax. However, upon heating or the addition of a solvent, the sequestered reagent(s) is/are released from the carrier wax and allowed to react with other soluble reagents, leading to the initiation of the PCR reaction. After the amplification is complete, the reactions are cooled to ambient temperature. Thus, a problem with these wax methods, however, is that the wax hardens after the completion of the amplification which makes sample recovery extremely tedious, since the wax tends to plug the pipet tips used to remove the sample. This is true even if the samples are reheated to melt the wax. Another potential problem is cross-contamination if tweezers are used to add wax beads, since slight contact between the tweezers and the tube caps can move DNA template between samples before the PCR reactions start. Furthermore, the addition of a wax or a grease layer can negatively affect a PCR reaction since increasing the total mass of the PCR reaction tube decreases the speed with which the contents of the tube approach the targeted temperatures in the thermal cycler.
Another way to implement a hot start PCR is to use DNA polymerase which is inactivated chemically but reversibly, such as AMPLITAQ GOLD(trademark) DNA polymerase. This enzyme preparation, distributed by PE Applied Biosystems, is distributed to users in inactivated form, but is reactivatable by heating. The required reactivation conditions, however, are extremely harsh to the template DNA: ten minutes at 95xc2x0 C. and at a nominal pH of 8.3 or lower results in reactivation of some 30% of the enzyme which is enough to start the PCR. See Moretti, et al., Biotechniques 25: 716-722 (1998). Because this treatment depurinates DNA every thousand bases or so, this enzyme can not be used to amplify DNA more than a few kilobases in length. Accordingly, the use of this enzyme is most efficient when it is restricted to amplifying target DNA with a length of approximately 200 base pairs.
An additional way of implementing a hot start is to combine the Taq DNA polymerase enzyme with a Taq antibody before adding it to the reagent. This method employs a monoclonal, inactivating antibody raised against Taq DNA polymerase. See Scalice et al., J. Immun. Methods, 172: 147-163 (1994); Sharkey et al., Bio/Technology, 12:506-509 (1994); Kellogg et al., Biotechniques, 16: 1134-1137 (1994). The antibody inhibits the polymerase activity at ambient temperature but is inactivated by heat denaturation. Unfortunately, the antibodies currently available for use in this method are not very efficient, and a 5 to 10-fold molar excess must be used to effect the advantages of a hot start PCR. For Klentaq-278, an amino-terminally deleted Thermus aquaticus DNA polymerase that starts with codon 279 which must be used at higher protein levels for long PCR (up to ten times more protein than Taq DNA polymerase), the levels of antibody necessary for a hot start become extremely high and the denatured antibody protein retains some inhibition for longer PCR targets. The original developer of anti-Taq antibodies (Kodak, now Johnson and Johnson) uses a triple-monoclonal antibody mixture which is more effective but is not commercially available and has not been tested in long PCR.
These methods used for hot starts require inclusion of an often expensive component (e.g., anti-Taq antibody) in the reaction mix and may place some undesirable constraints on the performance of the PCR such as a relatively short time period between when a reagent is prepared and when it must be used, or a lower efficiency of amplification.
Yet another method used for hot start PCR is to specially design primers with secondary structures that prevent the primers from annealing until cycling temperatures denature them. See Ailenberg et al., Biotechniques, 29: 1018-1020, 1022-1024 (2000). These specially designed primers are usually longer in length and special care must be taken in primer design. It may be inconvenient, expensive, or otherwise infeasible to design such primers.
Besides the grease/wax method, a low tech, inexpensive option of a physical hot start is to add the enzyme, the magnesium and/or the dNTPs to the reactions after they have heated to a temperature sufficient to ensure specificity of primer annealing. This xe2x80x9cmanualxe2x80x9d hot start method, besides being tedious and prone to error, commonly results in contamination and cross-contamination of PCR samples as the reaction tubes must be opened in the thermal cycler while they are hot.
Some PCR users believe they are performing a hot start when they set up PCR reactions in tubes on ice, then add the tubes to a thermal cycler block pre-warmed to 95xc2x0 C. Although some benefit arises from this method, the addition of only a few nucleotides to a primer can take place every second during the fifteen seconds or more that the tubes warm from 0xc2x0 to 25xc2x0 C. This is enough to initiate unwanted competitive PCR for reactions that require a hot start. Also, if many tubes are involved in an experiment, the tubes placed in the block first are heated for a longer time period at 95xc2x0 C. compared to the tubes placed later in the heating block thus resulting in a lack of reproducibility between samples.
Therefore, the current methods of hot start PCR are associated with multiple shortcomings. In cases of applying physical methods of the hot start, the possible problems include the ease of contamination, plugging up of pipet tips with wax or grease, and increase in time needed to reach target temperatures. In cases of applying chemical/biochemical methods of the hot start, the major drawbacks include the damage to template DNA resulting from harsh conditions needed to activate a chemically inactivated DNA polymerase, the excessive amounts of anti-Amplitaq antibody needed for inactivation of a DNA polymerase prior to initiation of a PCR reaction, and significant costs of obtaining commercially available antibodies. Furthermore, the use of specially designed primers may place unnecessary constraints on PCR reactions.
Accordingly, a need exists for obtaining novel or modified methods of xe2x80x9chot startxe2x80x9d PCR that would still provide all advantages of this procedure and at the same time minimize or completely eliminate some of its shortcomings.
Among the several aspects of the invention, therefore, may be noted the provision of novel processes for performing hot start PCR reactions. Briefly, the present invention is directed to processes for synthesizing nucleic acid extension products and specifically, to methods for amplifying a target nucleic acid sequence using PCR. Accordingly, the present invention provides reagents and kits which can be used to synthesize a nucleic acid extension product.
As such, it is an aspect of the present invention to increase the specificity of PCR product amplification by providing a new method for hot start PCR. In particular, the processes comprise sequestering magnesium ions in a precipitate thereby rendering the DNA polymerase inactive until the magnesium ions are released. In one aspect, the processes of the present invention utilize a reagent which comprises a precipitate containing magnesium. Alternatively, the precipitate is formed by combining a source of magnesium ions and a source of phosphate ions at a temperature of 4xc2x0 to 30xc2x0 C. The precipitate is combined with the PCR reaction components e.g., a thermostable DNA polymerase, deoxyribonucleoside triphosphates, a set of primers and a target nucleic acid sequence. The magnesium ions are then released from the precipitate, preferably by heating the mixture to a temperature sufficient to release the magnesium ions from the precipitate and into the mixture. The release of magnesium ions into the mixture activates the DNA polymerase thus allowing the extension of each primer to proceed.
A further aspect of the present invention is to provide kits for amplifying a target nucleic acid. In one embodiment, kits of the present invention comprise a container containing a source of magnesium ions and a container containing a source of phosphate ions which form a precipitate containing magnesium when combined at a temperature of 4xc2x0 to 30xc2x0 C., and instructions for amplifying the target nucleic acid. In another embodiment, the kits comprise a container containing a reagent comprising a precipitate containing magnesium and instructions for using the precipitate containing magnesium to amplify the target nucleic acid sequence. Preferably, other reaction reagents such as a DNA polymerase or a mixture of DNA polymerases and deoxyribonucleoside triphosphates are included in the kits of the present invention.
Other aspects and features will be in part apparent and in part pointed out hereinafter.